Evaluation and Application of Immunochemical Methods for

Chapter 23

Evaluation and Application of Immunochemical Methods for Mycotoxins in Food 1

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Downloaded by NORTH CAROLINA STATE UNIV on October 14, 2012 | http://pubs.acs.org Publication Date: March 23, 1995 | doi: 10.1021/bk-1995-0586.ch023

Mary W. Trucksess and Donald E. Koeltzow l

Division of Natural Products, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, DC 20204 Federal Grains Inspection Service, U.S. Department of Agriculture, Kansas City, KS 64153 2

Immunoassays have been developed for several mycotoxins including aflatoxins, deoxynivalenol, zearalenone, and fumonisins. These assays can determine such analytes in a variety of matrices and provide rapid analyses of a large number of test samples. Commercial immunochemical test kits are often evaluated on the basis of sensitivity, specificity, reproducibility, cost, time stability, and ease of use. Laboratory quality assurance checks, such as standard curves and positive controls, are essential. It is also important to differentiate interferences due to matrix effects from high levels of analyte. The criteria for interpretation of results from yes/no tests and quantitative tests are presented, as well as results of collaborative studies of immunochemical methods for aflatoxins and zearalenone in grains and grain products and surveillance findings obtained by using these methods for aflatoxins, deoxynivalenol, zearalenone, and fumonisins. Some of the criteria used to evaluate mycotoxin immunoassay procedures and experience in using them as surveillance tools can serve as models for similar approaches to the determination of pesticide residues in foods. Mycotoxins are toxic secondary metabolites produced by fungi in agricultural commodities. The fungi can invade grains growing in the field and during storage. Their ability to parasitize the plants depends on many factors, such as density of inoculum, plant species, prevailing temperature, tissue damage, insect activity, moisture, and harvesting practices. Many fungi produce mycotoxins, some of which are toxic to humans and animals. The mycotoxins found in significant quantities in naturally contaminated foods and feeds include the aflatoxins, deoxynivalenol (DON), zearalenone, and the fumonisins. These toxins can be present in corn, peanuts, cottonseed, tree nuts, cereal grains (wheat, barley, rice, oats), and many other commodities. Aflatoxins, a group of structurally related This chapter not subject to U.S. copyright Published 1995 American Chemical Society

In Immunoanalysis of Agrochemicals; Nelson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

TRUCKSESS & K O E L T Z O W

23.

Immunochemical Methods for Mycotoxins

327

mycotoxins identified in the early 1960s, are well known for their acute and chronic toxicity in animals and humans (I). The major aflatoxins of concern are B B2, G G , and M . DON is also known as vomitoxin from its effect on swine (2). Zearalenone is an estrogenic metabolite; it causes feed refusal and hyperestrogenism in swine (3). The fumonisins, a new class of mycotoxins, were isolated and characterized in 1988. They are produced primarily by Fusarium moniliforme and F. prolifération. F. moniliforme is the most common mold found on corn in the United States. Currently, seven fumonisins have been identified: FB^ FB , FB3, FB , FAi, FA , and F Q (4,5). FB causes brain damage in horses and lung edema in pigs. Fumonisins have been suggested as the possible cause of human esophageal cancer in South Africa (6). 1?

u

2

x


2

4

2

l

Analysis for Mycotoxins Analysis for mycotoxins is essential to minimize the consumption of contaminated food and feed. The problem is not simple. Deterrnining the concentrations of toxins in grains at the parts-per-billion levels required for the most important mycotoxins is difficult. A systematic approach is necessary. The approach generally followed consists of obtaining a relatively large sample, reducing it in bulk and particle size to a manageable quantity, and finally performing the analysis. Sampling commodities for aflatoxin contamination follows the U.S. Department of Agriculture (USDA) recommendations. Laboratory samples of at least 5-25 kg of corn, milo, and other grains are collected (7). To prepare a representative test portion for analysis, the laboratory sample is ground and mixed, so that the concentration of toxin in the test portion is the same as in the original laboratory sample collected. For pelletized feed a 1 kg laboratory sample is adequate, because the mycotoxins in the individual contaminated ingredients have presumably been uniformly distributed during feed manufacture. Similarly, a smaller laboratory sample is adequate for processed, comminuted, and mixed foods. Selection of Methods. For analysis we use authentic toxin standards and available methods, selecting appropriate methods for particular needs. The following criteria should be considered in selecting a method: number of analyses, time, location, cost of equipment, safety, waste disposal, and availability of experienced analysts. The new antibody-based immunochemical methods are simple, specific, and sensitive. Because mycotoxins are low molecular weight compounds, they do not independently induce an immune response when injected into laboratory animals; in most cases, they must first be derivatized and then conjugated to a carrier protein. The preparation of a suitable immunogen is an important step in the production of a specific antibody to a mycotoxin and in the development of an immunoassay.


328

IMMUNOANALYSIS OF AGROCHEMICALS


Iinmunochemical Methods Three major immunochemical techniques have been developed for mycotoxin deterrnination: radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (8). Thefirsttwo methods are based on competition between a free mycotoxin and a labeled mycotoxin for an antibody binding site. In the immunoaffinity column assay the antibodies are bound covalently to beaded agarose. The affinity column is used to bind the analyte to the antibody-agarose packing and serves as a concentration tool for the analyte. After elution, the analyte is subjected to further procedures. At present the ELISA and the immunoaffinity column assay techniques are more commonly used than the RIA. Solid foods are typically extracted with aqueous methanol and diluted with water before analysis to maintain the native protein structure of the antibody and enzyme conjugate. Commercial Immunoassay Kits. Many commercial immunoassay kits for mycotoxins in agricultural products are being marketed. These kits provide the reagents, materials, and instructions necessary to perform the tests. A list of some of the manufacturers and types of immunoassay kits is found in Table I. The kits are intended for rapid qualitative identification or quantitative determination. This listing includes only those applicable to mycotoxins for which our laboratory has received descriptive information, and is not intended to be all-inclusive. Criteria for Evaluation of Commercial Immunoassay Kits At present there are no standard criteria for evaluating commercial immunoassay kits. Several organizations have been actively engaged in developing evaluation guidelines: Association of Official Analytical Chemists International (AOACI), U.S. Environmental Protection Agency (EPA), U.S. Department of Agriculture (USDA), and U.S. Food and Drug Adniinistration (FDA). Table Π summarizes some of the criteria used by the USDA-FGIS (Federal Grains Inspection Service) to evaluate immunoassays for aflatoxin. Initial Evaluation. In FDA laboratories, requested information obtained from manufacturers is evaluated with emphasis on sensitivity, applicability, stability, clarity of instructions, quality control, comparison with reference method (if possible), cost, and equipment. An initial choice is made after this review is completed. Kits are purchased and critically evaluated. A slightly different approach is usai in FGIS laboratories. A notice is published in the Federal Register requesting that all manufacturers of test kits capable of detecting the analyte of interest submit information on test kit capabilities. These data are reviewed according to criteria similar to those described for FDA laboratories. Test kits representing each of the different analytical methods available (well, cup, column, etc.) are examined in the laboratory to provide experience in using the various test methodologies. Kits that meet the basic FGIStimelinessand safety requirements shown in Table Π are purchased and collaboratively tested in several FGIS field inspection laboratories.



Environ. Diag. Sys. Neogen Neogen Vicam Environ. Diag. Sys. Neogen

Deoxynivalenol

Fumonisins

Zearalenone

card well

well column

card well

Table I. Commercially Available Mycotoxin Immunoassay Kits Type Company card Environ. Diag. Sys. Aflatoxins well, probe Idexx well Idetak cup Inter. Diag. Sys. well Neogen column Vicam

Mycotoxin



330

IMMUNOANALYSIS OF AGROCHEMICALS

Table Π. Design Criteria and Test Performance Specifications for Quantitative Aflat oxin Test Kits

1. 2. 3. 4.

Time required for completion: 30 min. Capability of analyzing for B , G and G . Applicability: corn, corn meal, etc. Acceptable accuracy, precision limits. 2

Aflatoxins added ng/g 0 10 20 30 320

5. 6. 7. 8. 9. 10.

u

2

Accuracy ng/g

Precision ng/g

«S7.0 ±8.0 ±10.0 ±12.0 ±60.0

4.0 6.0 8.0 10.0 32.0

Should not include toxic solvents and reagents. Comparative accuracy of test kits on corn samples naturally contaminated with aflatoxins at about 20 and 100 ng/g. The limit of detection of test kits,

Evaluation and Application of Immunochemical Methods for

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